Method for isolating a thiol
A method of detecting a member of the taxa actinomycetes is provided. A method also is provided for detecting mycothiol or precursor thereof. An antibody is provided when binds to mycothiol or a mycothiol precursor. A method is further provided for diagnosis of a subject having or at risk of having an actinomycetes-associated disorder. A method is also provided for identifying a sample with altered production of mycothiol or a precursor thereof. A method is provided for detecting mycothiol or precursor thereof in a bacterial colony. Kits are also disclosed which are useful for detecting the presence of mycothiol or precursor thereof in a sample.
1. A method of isolating a thiol R′SH from a thiol-containing mixture, the method including the steps of
forming a mixed disulphide R′SSR of the thiol R′SH by reacting the thiol R′SH with a second mixed disulphide R″SSR, in which R is a hydrophobic moiety which is not immobilised on a stationary phase, and R″ is selected so that a forward reaction of the R′SH with the second mixed disulphide R″SSR to form R′SSR and R″SH is favoured over a reverse reaction of R″SH with R′SSR back to R″SSR and R′SH;
purifying the mixed disulphide R′SSR by a process selected from selective precipitation and chromatography;
reacting the purified mixed disulphide R′SSR with a reducing agent to produce the thiol-containing mixture of thiols R′SH and RSH; and
separating the mixture of thiols R′SH and RSH to isolate the thiol R′SH, wherein R′SH is 1-D-myo-inosityl-2-deoxy-2-(N-acetyl-L-cysteinyl)amino-α-D-glucopyranoside or mycothiol, the second mixed disulphide R″SSR is 2-thiopyridyl-6-hydroxynaphthyldisulphide, and R″ is a 2-thiopyridyl group.
2. The method as claimed in claim 1 , wherein purifying the mixed disulphide R′SSR includes exploiting an increased hydrophobicity thereof relative to the thiol R′SH.
3. The method as claimed in claim 1 , wherein the mixed disulphide is purified by means of reversed phase high performance liquid chromatography (HPLC).
4. The method as claimed in claim 1 , wherein the reducing agent is selected from a group consisting of dithiothreitol and β-mercaptoethanol.
5. The method as claimed in claim 1 , comprising separating the mixture of thiols R′SH and RSH by high performance liquid chromatography (HPLC).
6. The method as claimed in claim 5 , wherein the high performance liquid chromatography is performed on a C18 reversed phase medium.
7. The method as claimed in claim 1 , wherein the mixed disulphide R′SSR is 2-S-(mycothiolyl)-6-hydroxynaphthalenedisulphide.